Photobiology of Rhodopsin and the Cone Pigments

描述（由申请人提供）：视锥细胞负责明视，即正常光照条件下的视觉过程。视锥细胞感受器必须在很宽的光强度范围内工作，并覆盖整个可见光谱范围。在这些不同条件下发挥作用的能力主要是由于高度优化的GPCR光转导蛋白质，非正式地称为锥色素。这些蛋白质具有范围从350至660 nm的吸收最大值，并且在吸收光时，经历有效的光漂白序列以产生活化的蛋白质。随后，转导素与活化的蛋白质结合，产生神经冲动和视觉。在之前NIH资助的研究中，一个关键的观察结果是锥状色素在光活化过程中经历了一个电荷转换。本研究的一个主要目的是探索是否在红色和蓝色锥色素中也存在一种反相开关机制，如果是，则描述分子细节。为了实现这一目标，我们将使用振动和电子光谱在温度从10 K到环境的陷阱和特征的光漂白中间体。将使用定点诱变来鉴定负责波长选择的关键残基和互补开关的性质。从同源性研究中可以清楚地看出，许多红色视锥细胞在性质和反向开关的实现上不同于绿色、蓝色和UV视锥细胞。事实上，红色视锥细胞可能完全没有这种机械特征。本研究的另一个目的是系统地确定在紫外线，蓝色，绿色和红色锥的波长选择的机制。虽然我们的研究确定了蓝色和紫外光锥中波长选择的关键特征，但仍有许多问题有待理解。我们在这项研究中纳入红色视锥细胞是新的，我们对这个话题的热情部分取决于我们相信红色视锥细胞是根本不同的。我们有初步的证据，在我们的初步研究讨论，深红色锥使用至少一个新的机制，涉及操纵发色团环构象的波长选择。独特的波长选择和显著不同（或不存在）的反向转换机制的组合使红锥成为重要的目标。我们的研究将包括使用分子轨道理论来探测锥色素的结构-功能关系，并计算结合发色团的光谱性质。我们将重构我们的MNDO-PSDCI代码并改进接口，使这些程序对科学界更有用。和以前一样，我们将免费向感兴趣的研究人员提供这些程序。公共卫生相关性：人们越来越需要了解光漂白和恢复机制与光暴露在锥光感受器。因为这些细胞对于人类的明视是必不可少的，所以了解相关的光转换色素的结构和功能关系是很重要的。这项研究的项目目标可能有助于了解黄斑疾病，这涉及视锥细胞的损失，视锥营养不良和其他眼部疾病，这涉及视网膜光感受器的损伤或功能减弱。

DESCRIPTION (provided by applicant): Cone cells are responsible for photopic vision, the visual process under normal light conditions. The cone receptors must operate over a wide range of light intensities and cover the full range of the visible spectrum. The ability to function under these diverse conditions is due primarily to the highly optimized GPCR light-transducing proteins informally called cone pigments. These proteins have absorption maxima that range from 350 to 660 nm, and upon the absorption of light, undergo an efficient photobleaching sequence to produce an activated protein. Subsequent binding of transducin to the activated protein results in a nerve impulse and vision. A key observation made during the previous NIH funded study was that cone pigments undergo a counterion switch during photoactivation. A key aim of this study is to explore whether a counterion switch mechanism is also active in the red and blue cone pigments, and if so, to characterize the molecular details. To achieve this goal, we will use vibrational and electronic spectroscopy at temperatures from 10K to ambient to trap and characterize the photobleaching intermediates. Site directed mutagenesis will be used to identify the key residues responsible for wavelength selection and the nature of the counterion switch. It is clear from homology studies that many of the red cones differ from the green, blue and UV cones in nature and implementation of the counterion switch. Indeed, it is possible that the red cones lack this mechanistic feature entirely. An additional aim of this study is to systematically identify the mechanisms of wavelength selection in the UV, blue, green and red cones. Although our research identified key features of wavelength selection in the blue and UV cones, much remains to be understood. Our inclusion of the red cones in this study is new, and our enthusiasm for this topic rests in part on our belief that the red cones are fundamentally different. We have preliminary evidence, presented in our preliminary studies discussion, that the deep red cones use at least one new mechanism for wavelength selection involving manipulation of the chromophore ring conformation. The combination of unique wavelength selection and a significantly different (or absent) counterion switching mechanism make the red cones an important target. Our studies will include the use of molecular orbital theory to probe structure-function relationships in the cone pigments, and to calculate the spectroscopic properties of the bound chromophores. We will refactor our MNDO-PSDCI code and improve the interface to make these procedures more useful to the scientific community. As before, we will provide these procedures to interested researchers without charge. PUBLIC HEALTH RELEVANCE: There is a growing need to understand the photobleaching and recovery mechanisms associated with light exposure in cone photoreceptors. Because these cells are essential for human photopic vision, it is important to understand the structure and function relationships in the associated light transducing pigments. The project goals of this research may help understand macular disease, which involves loss of cone cells, cone dystrophy, and other eye diseases, which involve damage or diminished function of retinal photoreceptors.